Optochemical sensors (optodes), in the following referred to as "optical sensors" or "sensors" for reasons of simplicity, these days are widely employed, preferably in the form of membranes, in sensor configurations in order to quantify particular substances such as, e.g., oxygen or glucose in a sample. Optical sensors are used, for instance, in environmental measuring technology and in emergency medicine (blood gas analysis). The mode of functioning of optical sensors and the basic structure of a sensor configuration generally comprising an optical sensor formed of several layers, an excitation light source and an optoelectronic detection system has been described in the literature (e.g., Sensors and Actuators B 11 (1993), pp. 281-289; Sensors and Actuators B 29 (1995), pp. 169-173).
There has already been known a plurality of indicator substances and sensors which respond to the chemical substances mentioned and, in particular, oxygen by changing an optical characteristic of the indicator. In U.S. Pat. No. 3,612,866, for instance, Stevens describes an optical sensor capable of being calibrated and sensitive to oxygen, which contains the dye pyrene, the luminescence of which is extinguished by diffusing-in oxygen in a concentration-dependent manner. At the same time, the sensor includes a reference sensor on a neighboring site, which reference sensor is masked by an additional oxygen-impermeable membrane. The concentration of oxygen is determined by the ratio of the signals of the two areas.
Furthermore, Luibbers in U.S. Reissue Pat. No. RE 31,879 describes a luminescence-optical oxygen sensor including the indicator pyrene butyric acid, optionally stirred into a silicone matrix having a high permeability for oxygen, whose sensitive layer is embedded between a light-permeable covering layer and an oxygen-permeable base layer contacted by the analyzed liquid.
In another U.S. Pat. No. 4,657,736 Marsoner describes an oxygen sensor comprising modified dyes that are readily soluble in silicone, thus offering an enhanced stability of the sensor against the aggregation of dye molecules. The sensor is prepared by stirring the dye into a prepolymer and subsequent polymerization to silicone.
In U.S. Pat. No. 4,752,115 Murray describes an oxygen-sensitive layer of a transition metal complex in a plasticized organic polymer matrix (e.g., PVC), which is applied as a layer onto a fiber optic conductor element via which the excitation light is launched. Those complexes, in general, are more photostable than organic dyes. Again, the luminescence intensity is measured as a function of the concentration of oxygen in the layer.
In U.S. Pat. No. 4,775,514 Barnikol describes a luminescent surface for determining oxygen in gases, liquids and tissues. The sensitive layer on the surface is comprised of a homogenous mixture of an organic dye (pyrene, coronene, etc.) with silicone.
Khalil in U.S. Pat. Nos. 4,810,655 and 5,043,286 describes measurements of the decay times of phosphorescent dyes having long decay times readily accessible by measuring techniques, instead of luminescence intensity measurements. The fluorinated porphyrins used exhibit relatively high photostabilities. In addition, the parameter decay time is less prone to photodecomposition effects as compared to luminescence intensity.
The same technique is employed by Bacon in U.S. Pat. No. 5,030,420, which describes an oxygen sensor comprised of a ruthenium(II) complex immobilized in a silicone that is impermeable to many liquids such as, e.g., acids and bases, complexing agents, oxidizing and reducing liquids, yet is highly permeable for oxygen and other gases. However, that sensor contains the indicator electrostatically bound to filler particles (silica gel) in the silicone, a fact the author's attention is drawn to only at a later point of time (e.g., Sacksteder, et al., Anal. Chem. 65, p. 3480). This provides for a good stability against washing out of the dye in the first place.
The stability of a sensor against washing out of the indicator also is the topic of proposals in U.S. Pat. No. 5,070,158 to Holloway and U.S. Pat. No. 5,128,102 to Kaneko, which disclose the possibility of chemically binding indicator molecules to a polymer matrix.
Another way of improving the stability of a sensor against the loss of its indicator and hence the deterioration of the photophysical properties of the membrane is set forth by
Markle in U.S. Pat. No. 5,511,547. A special silicone matrix comprising polar carbinol groups serves to enhance the interaction between indicator (e.g., tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chloride) and matrix in order to reduce the washing out, and also the aggregation, of the indicator molecules. Those measures are, however, not suitable for substantially enhancing the photostability of the membrane per se.
Finally, Jensen in U.S. Pat. No. 5,242,835 describes a method for determining the concentration of oxygen in a sample by detecting the emission of the singlet oxygen itself, which is excited by energy transmission during the extinction of the luminescence, occuring at a wavelength of approximately 1270 nm. Also that method is prone to photodecomposition of the indicator or the matrix by exactly that reactive singlet oxygen, the latter returning into its ground state without radiation during a photochemical reaction, thus causing also the sensitizer molecules (indicators) serving the production of the singlet oxygen to be attacked.
As already mentioned, optochemical sensors, in general, contain dyes which respond to a change in the concentration of the substance to be analyzed within the sensitive layer by changing their photophysical properties. The intensity and decay time of the luminescence of oxygen sensors will, for instance, decrease with the concentration of oxygen increasing in the sensitive layer.
A problem faced by many sensors and, in particular, oxygen sensors is their proneness to decomposition, which is triggered by the irradiated excitation light (Anal. Chem. 1991, 63, pp. 337-342). Both the luminescence indicator itself and the matrix are susceptible to decomposition. The problem of photoinduced decomposition occurs, in particular, if radiation occurs at a high intensity in order to enhance the signal quality and reduce the measuring error or if a single sensor is operated over a very long period of time as happens in the monitoring of chemical substances. Since the intensity of luminescence is a direct function of the concentration of dyes, the photodecomposition of these dyes in sensors of that kind is undesired.
The decay time of the luminescence of a sensor with dyes immobilized therein is a function of the concentration of the luminophores, in particular, if and when
firstly, an overlay background luminescence (of sources other than the dye) occurs, PA1 secondly, luminescent degradation products are again formed by the photodecomposition of the dyes, the decay time of which degradation products differs from the decay time of the luminescence of the starting dyes, and PA1 thirdly, a decomposition of the matrix (environment) of the dye changes the photophysical properties of the latter.
Thus, the decomposition of the luminophore or of the matrix (for instance, by the action of singlet oxygen) of typical sensor systems is undesirable for the following reasons:
On the one hand, the mere decrease of the luminescence intensity (at a constant decay time) results in a relative increase of the background fluorescence and hence in a change of the calibration curves. The decrease of the luminescence intensity constitutes a problem to all sensor systems if the radiation quantity to be measured is thereby lowered to such an extent that the dynamic range of the receiver electronics is left. A decrease of the luminescence intensity causes particular problems to sensor systems based on the measurement of the luminescence intensity, but also to measuring systems evaluating a phase shift of the luminescence, which is caused by the finite life time of the excited states of the dye molecules.
On the other hand, a change in the luminescence decay time by photodecomposition directly leads to a change of the photophysical properties of the sensor system, this having adverse effects both on intensity measuring systems and on decay time measuring systems.
As already described above, optochemical sensors will change their photophysical properties (luminescence intensity, quantum efficiency, decay time, . . . ) in the presence of substances to be analyzed ("quencher"). The function between the concentration of the substance and the photophysical parameter, i.e., the calibration function, for instance, has the form of a Stern-Volmer equation (1), (3): EQU I.sub.0/ I=1+K.sub.SV [Q] (1a) EQU .tau..sub.0 /.tau.=1+K.sub.SV [Q] (1b) EQU K.sub.SV =k.sub.q.tau..sub.0 (1c)
K.sub.SV is the Stern-Volmer constant, k.sub.q is the bimolecular deactivation rate and [Q ] is the concentration of the quencher. I is the luminescence intensity and .tau. its decay time. The index 0 indicates the absence of a quencher.
In many cases, the decay function of the luminescence may be described by a multi-exponential model (Equ. 2). ##EQU1##
i(t) being the time course of the emission after a comparatively short excitation pulse. B.sub.i are the amplitudes. .tau..sub.i are the time constants and m is the number of monoexponential model functions (index i), the sum of which may be described as the decay function.
In this connection, a more complex form of the Stern-Volmer equation will apply: ##EQU2##
f.sub.0i being the relative portion of the i-th decay time component. .tau..sub.av is a mean decay time, weighted according to the amplitudes B.sub.i. The special case m=2 and K.sub.SV2 =0 is designated as a stray light model, wherein stray light may be constituted, for instance, by background fluorescence. Hence follows that the luminescence intensity and luminescence decay time must lead to a change in the parameter values of the calibration function through influences other than the present quencher as well as by a change in the stray light portion (background fluorescence).
The intensity I of luminescence is directly proportional to its quantum efficiency .PHI..sub.L : EQU I=k..PHI..sub.L (4)
k being a proportionality constant.
The quantum efficiency, in turn, is related to the decay time via the radiating deactivation rate k.sub.r, of the excited state: EQU .PHI..sub.L =k.sub.r.tau..sub.0 (5)
From the above functions it is apparent that a change in the decay time results directly in a change of the luminescence intensity (Equ. 4 and Equ. 5) and of the Stern-Volmer constant (Equ. 1c). Thus, the characteristic parameters, which are collected, for instance, by way of the calibration of a sensor by means of test substances of known compositions, will be changed, calling for the recalibration of the sensor. Frequent recalibrations, however, involve immense drawbacks if measuring is to be effected over extended period of times (monitoring). In addition, the photophysical properties of a sensor of this type may change in a manner that it will no longer offer the sensitivity required for carrying out measurements.
Such a sensor system, in general, is of the widely known optical configuration schematically represented in FIG. 1. That sensor system comprises: a light source 1 for excitation light of a suitable wavelength (matching with the absorption spectrum of the luminescence dye), a detector 2 for detecting the luminescence of the sensor 3, which is comprised of a sensitive layer 4 containing the luminescence indicator, an optical insulation layer 5 and a transparent carrier 6, filters 7, 8 for the excitation light and the emitted light, respectively, a beam splitter 9 and a measuring cell 10, which may, for instance, be a flow tube through which the sample to be assayed is transported. The transport direction of the sample is symbolized by an arrow in FIG. 1.
Measuring is effected in the following manner:
The sample to be analyzed such as, e.g., blood in which the oxygen concentration is to be determined, is transported through the flow tube 10, getting into contact with the optical insulation layer 5, which is permeable to oxygen. The oxygen gets through the optical insulation layer 5 into the sensitive layer 4, which constitutes a matrix of, for instance, a polymer containing the luminescence indicator for oxygen. The luminescence indicator is excited to luminescence by excitation light 11 coming from the light source 1, said luminescence being quenched by oxygen in a concentration-dependent manner. The emission 12 is electronically detected in the detector 2, the oxygen concentration in the sample being calculated from that value.
A light source with a time-related modulation of its intensity (pulse operation, sinus or rectangular modulation) and a detector resolving in time, or modulated in terms of sensitivity, may be used for measuring the decay time or the phase shift. In the simplest case, the phase shift .DELTA..PHI. relative to the sinusoidally modulated light source is related to the decay time .tau. via the circular frequency .omega. of the sinus modulation: EQU tg(.DELTA..PHI.)=.omega..tau. (6)
Thus, also the phase shift of the sensor will change if the photodecomposition products have decay times that differ from that of the starting dye or if the environment (matrix) of the dye is influenced in a manner that the decay time changes.
All of the sensors hitherto described in the scientific literature and in the patent literature are not suitable for substantially reducing the above-mentioned photodecomposition effects. It is true that some of the above-mentioned systems are explicitly described as systems of enhanced stability, yet this may be realized to a very limited degree only by traditional measures such as the selection of a particular polymer matrix or immobilization method.
Carraway, et al. have described (Anal. Chem., 1991, 63, pp. 337-342) that the photochemical decomposition of the sensor is promoted by oxygen in oxygen sensors, but that singlet oxygen, which is formed by quenching of the excited luminescence indicator, apparently does not contribute directly to sensor decomposition and apparently is not the main cause of sensor decomposition. To stabilize oxygen sensors, those authors suggest to photolyze the sensor prior to its use in order to destroy reactive components supposed to be responsible for decomposition. Yet, also that measure is not suitable for preventing photoinduced decomposition.